Methods and reagents for quantitation of cell-surface molecule expression on peripheral blood cells

ABSTRACT

Improved methods, reagents, and kits for quantitation of HLA-DR and/or CD11b expression on peripheral blood cells are presented. Inclusion of a lysosomotropic amine, such as chloroquine, during staining stabilizes HLA-DR and CD11b expression. Use of a novel anti-CD14 conjugate, anti-CD14-PerCP/CY5.5, permits the ready discrimination of monocytes. The improved methods, reagents, and kits may be used to assess immune competence, and to direct and monitor immunostimulatory therapies in septic patients exhibiting monocyte deactivation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation U.S. patent application Ser. No.09/406,013, filed Sep. 24, 1999, which issued as U.S. Pat. No.6,423,505, which is a continuation-in-part of U.S. patent applicationSer. No. 09/204,860, filed Dec. 3, 1998, which issued as U.S. Pat. No.6,200,766, the disclosures of both of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to improved methods, reagents and kits forquantitating the expression of HLA-DR and/or CD11b on the surface ofhuman peripheral blood cells, particularly on the surface of peripheralblood monocytes. The invention further relates to methods for assessingimmune competence and for directing and monitoring immunostimulatorytherapies for sepsis based upon the levels of monocyte HLA-DR expressionso measured.

BACKGROUND OF THE INVENTION

Sepsis is one of the most common causes of death in developed countries.Incidence is increasing, and mortality remains high.

Early efforts to understand sepsis and to intervene in the progressivemultiorgan failure of septic shock focused upon readily observablephysical, physiologic, and anatomic symptoms. To this day, the acutemanagement of fever, infection, coagulatory dysfunction, vascularcollapse and end-organ failure remains the standard of care.

More recent efforts, however, have focused upon immunologic mediatorsthought to underlie these systemic processes.

Animal models of sepsis have, for example, implicated a number ofcytokines as mediators of the systemic inflammatory response seen earlyin the septic patient. In these models, acute parenteral challenge withendotoxin or with gram negative bacteria leads to production of tumornecrosis factor α (TNFα), interleukin-1 (IL-1), and gamma interferon(IFNγ). Gamma interferon has been shown to act synergistically with TNFαin inducing shock in these animals.

Yet recent efforts to treat sepsis by immunomodulation have provendisappointing. Attempts to reduce or ablate the effects ofproinflammatory cytokines, particularly TNFα and IL-1, have not onlyfailed to improve outcome, but have in several cases increasedmortality. Fisher et al., JAMA 271:1836-43 (1994); Fisher et al., N.Engl. J. Med. 334:1697-1702 (1996); Fisher et al., Crit. Care Med.21:318-327 (1993); reviewed in Bone, JAMA 276:565 (1996). There thusexists a need for immunomodulatory therapies that improve clinicaloutcomes in sepsis.

Recently, several observations have motivated an alternative, seeminglycontrarian, immunomodulatory approach.

Acute bacterial invasion is in fact an atypical clinical presentationfor human sepsis. In most patients, sepsis is a late complication oftrauma, burn, or major surgery. Infections in these patients—cominglate, as a secondary response to antecedent injury, rather than early,as the primary and actual cause of septic shock—bespeak a possiblesystemic immunosuppression or immune paralysis, rather than a state ofhyperimmunity as predicted by the acute animal models.

In particular, studies have shown that HLA-DR expression by monocytes isseverely depressed after trauma, and that such depressed levelscorrelate clinically with an increased susceptibility of trauma patientsto infection. Polk et al., Ann. Surg. 204:282 (1986); Hershman et al.,Clin. Exp. Immunol. 77:67-70 (1989).

Depression of monocytic HLA-DR expression has also been shown in a studyof patients undergoing elective or emergent neurosurgery: patients whodevelop infectious complications in the postoperative period display asignificantly lower level of monocytic HLA-DR expression than patientswith an uncomplicated course; very low HLA-DR expression (fewer than 30percent of peripheral blood monocytes positive for HLA-DR expression)predicts high risk for infection following surgery. Asadullah et al.,Crit. Care. Med. 23:1976-1983 (1995).

Depression in HLA-DR expression has further been observed in theperipheral blood monocytes of septic patients with a wide variety ofprecipitating ailments. In these latter studies, surfaceimmunophenotypic changes were further associated with decreasedmonocytic antigen-presenting function, reduced production of TNFα, IL-1and IL6, anergy, and alterations in lymphocyte activity. Volk et al.,Behring Inst. Mitt. 88:209-215 (1991); Döcke et al., in Reinhart et al.(eds.), Sepsis: Current Perspectives in Pathophysiology and Therapy, NewYork: Springer-Verlag (1994) pp 473-500.

Monocytes, like macrophages, B cells, and dendritic cells, are“professional” antigen presenting cells (APCs). Although a number ofcell types are capable of processing soluble antigens for subsequentdisplay to T lymphocytes, the so-called “nonprofessional” APCs lack theaccessory molecules required to complete the process of T cellactivation. “Professional” antigen-presenting cells, such as monocytes,not only process and present antigens in the context of MHC, but alsopossess the additional accessory molecules required to complete T cellactivation, rendering them critical to the development of a full Tcell-directed immune response. Reversal of monocytic deactivation inlate-stage sepsis might, therefore, be expected to improve immunefunction, conferring clinical benefit.

Gamma interferon (IFNγ) is a major activator of monocytes. Itupregulates the surface expression of costimulatory and HLA molecules,increasing monocyte antigen-presenting capacity, and primes for theLPS-induced production of proinflammatory cytokines. Young et al., J.Leukocyt. Biol. 58:373-381 (1995).

A single clinical trial of gamma interferon treatment of late-stagesepsis has been reported. Peripheral blood monocyte HLA-DR levels weremonitored in patients meeting the inclusion criteria for severe sepsis.Gamma interferon was administered to those patients in whom, over twoconsecutive days, fewer than 30% of peripheral blood monocytes measuredpositive for HLA-DR expression. Treatment was continued until thepercentage of monocytes with demonstrable HLA-DR expression remainedover 50% for three days. Of the 10 patients, 8 showed an increase inmonocyte HLA-DR expression within 1 day of treatment; the other 2responded within 2 to 3 days. The recovery of monocytic HLA-DRexpression was associated with restitution of monocytic function invivo, as evidenced by a significant increase of TNFα and IL-6 plasmalevels during treatment and a more favorable clinical outcome. Kox etal., Arch. Intern. Med. 157:389-393 (1997); Döcke et al., Nature Med.3:678-680 (1997).

Because administration of a proinflammatory cytokine would becontraindicated, however, in the early, hyperimmune phase of sepsis,there exists a need for a rapid, reliable method for measuring HLA-DRexpression on peripheral blood monocytes. There further exists a needfor a method that would report values for a given peripheral bloodsample that are reliable and substantially independent of the individualtesting laboratory.

Typically, as in the reported clinical study, monocyte HLA-DR expressionis assessed flow cytometrically. Monocytes are distinguished from otherperipheral blood cells by either surface immunophenotype, physicalproperties (side scatter and/or forward scatter), or some combinationthereof; HLA-DR levels are assessed on the monocytes so distinguished byuse of a fluorophore-conjugated anti-HLA-DR antibody.

Monocytes may, for example, be distinguished using an antibody specificfor CD14. CD14, a receptor for lipopolysaccharide, is expressedpredominantly on cells of the myelomonocytic lineage; in peripheralblood, CD14 is expressed principally by monocytes. But granulocytes inthe blood also react with anti-CD14 antibodies, albeit weakly, and withpresent anti-CD14 conjugates cannot be completely discriminated frommonocytes. Gating out CD14^(dim) cells, as a means of removinggranulocytes from the analysis, removes CD14^(dim) monocytes as well,confounding the HLA-DR analysis. There thus exists a need for afluorophore that, when conjugated to anti-CD14 antibody, would permitthe immunocytochemical discrimination of monocytes from granulocytes.

HLA-DR may readily be labeled on the surface of peripheral blood cells,including monocytes, using a fluorophore-conjugated anti-HLA-DRantibody. But the surface expression of HLA-DR on the surface ofmonocytes is not a simple, static, and stable phenotype. MHC restrictionimposes conflicting demands on the protein processing machinery of theAPC: on the one hand, there is a requirement forproteolytically-processed peptide antigen; on the other, there is arequirement for intact MHC class II protein. These concurrentrequirements are met by a finely choreographed, and as yet incompletelyunderstood coordinated movement of endocytosed antigen andnewly-synthesized MHC class II molecules through various internalcompartments of the cell. Cresswell, Annu. Rev. Immunol. 12:259-93(1994). Rapid recycling of class II molecules from the surface, throughcompartments in part distinct from those traversed by newly-synthesizedMHC, and then back to the surface, implicates yet other, likelyintersecting, intracellular pathways. Reid et al., Nature 346:655-657(1990); Roche et al., Proc. Natl. Acad. Sci. USA 90:8581-85 (1993);Watts, Annu. Rev. Immunol. 15:821-50 (1997).

The level of HLA-DR-specific fluorescence reported by peripheral bloodmonocytes depends upon the duration of incubation with anti-HLA-DRantibody, evidence of the dynamic nature of HLA-DR expression. This timedependence makes reliable absolute measurements of HLA-DR expressiondifficult. There thus exists a need for a method and reagents that wouldpermit the stabilization of HLA-DR levels for flow cytometric assay.

SUMMARY OF THE INVENTION

The present invention solves these and other problems in the art byproviding improved methods, reagents, and kits for quantitating theexpression of HLA-DR and/or CD11b on the surface of human peripheralblood cells, particularly on the surface of peripheral blood monocytes.

The invention is based, in part, upon the novel finding that includingchloroquine during staining of peripheral blood with anti-HLA antibodystabilizes the HLA-DR-specific signal obtained from CD14⁺ monocytes.This is surprising: chloroquine has been reported to be unable to blockthe cellular recycling of MHC glycoproteins, and other agents thataffect protein processing, such as Brefeldin A and monensin, have beenshown ineffective in stabilizing HLA-DR expression. Equally surprisingis the novel finding that including chloroquine during staining ofunfractionated peripheral blood with anti-CD11b antibody stabilizes theCD11b-specific signal.

The invention is also based in part upon the finding that conjugation ofan anti-CD14 antibody to a fluorescence energy resonance transfer tandemfluorophore, PerCP/CY5.5, provides a high intensity, highly uniform andclustered signal from peripheral blood monocytes, permitting the readydiscrimination of monocytes from granulocytes. Incorporating theseimprovements provides an assay well-suited for clinical use, permittingthe rapid and reliable quantitative measurement of peripheral monocyticHLA-DR levels.

In a first aspect, therefore, the present invention provides a method ofmeasuring HLA-DR expression on the surface of human blood cells,comprising contacting a sample containing human blood cells with alysosomotropic amine and an antibody specific for HLA-DR, and thendetecting the binding of the anti-HLA-DR antibody to the cells. Inpreferred embodiments, the lysosomotropic amine is chloroquine. Inpreferred embodiments, the anti-HLA-DR antibody is conjugated to afluorophore, preferably at a defined molar ratio; particularly preferredis conjugation to PE at a defined molar ratio, and especially preferredis conjugation to PE at a molar ratio of 1:1.

The invention also provides for the measurement of HLA-DR on the surfaceof peripheral blood monocytes. In preferred embodiments, amonocyte-distinguishing antibody, typically anti-CD14, is included inthe staining step. In particularly preferred embodiments, the anti-CD14antibody is conjugated to the PerCP moiety of a PerCP/CY5.5 tandemfluorophore molecule, permitting single-laser, multicolor flowcytometric analysis.

In preferred embodiments, a whole blood sample is employed, although themethods and reagents may equally be used with appropriate bloodfractions, such as fractions enriched in peripheral blood mononuclearcells. In the whole blood embodiments, a further step of lysing theerythrocytes in the blood sample preferably intervenes between thestaining step and the detection step. The method may include yet afurther step of removing lysis debris after the lysis step but beforedetection.

Results from the improved HLA-DR assay of the present inventioncorrelate well with results from prior, but problematic, assays. Thishigh degree of correlation permits the direct translation of theestablished clinical criteria to measurements reported by the presentassay.

Thus, in another aspect, the present invention provides a method ofassessing the immune status of a human patient, comprising the steps ofcontacting a sample containing the patient's blood cells with alysosomotropic amine and an antibody specific for HLA-DR; detecting thebinding of the anti-HLA-DR antibody to the monocytes in the sample; andthen comparing the level of binding so detected with that so detectedfrom human controls. In preferred embodiments of this method, thelysosomotropic amine is chloroquine, the anti-HLA-DR antibody isconjugated to PE, and a monocyte-distinguishing antibody, such asanti-CD14, preferably anti-CD14-PerCP/CY5.5, is included in the stainingstep.

The invention also provides, in a related aspect, a method fordetermining the suitability of immunostimulatory therapy in a patientwith sepsis, comprising contacting a sample containing the patient'sblood cells with a lysosomotropic amine and an antibody specific forHLA-DR; detecting the binding of the anti-HLA-DR antibody to themonocytes in the sample; and then comparing the level of binding sodetected with that detected from normal controls. Patients with levelsof binding less than that of controls are determined to be suitable forimmunostimulatory treatment. Applying present clinical criteria, sepsispatients averaging fewer than 5000 anti-HLA-DR antibodies per monocyteare determined to be suitable for immunostimulatory treatment, withpatients averaging fewer than 3000 anti-HLA-DR antibodies per monocytedetermined to be particularly suitable, and with patients averagingfewer than 3000 anti-HLA-DR antibodies per monocyte for two consecutivedays determined to be especially suitable for immunostimulatorytreatment.

In especially preferred embodiments of the method for determining thesuitability of treatment, a peripheral blood sample is in the first stepcontacted with an anti-HLA-DR PE antibody, an anti-CD14-PerCP/CY5.5antibody, and chloroquine.

In another aspect, the present invention provides compositions forperforming the subject methods. The compositions for flow cytometricmeasurement of HLA-DR on human peripheral blood cells comprise afluorophore-conjugated anti-HLA-DR antibody and a lysosomotropic amine,preferably chloroquine. The anti-HLA-DR antibody is preferablyconjugated to PE, most preferably at a defined molar ratio, and inespecially preferred embodiments, at a molar ratio of 1:1.

For flow cytometric measurement of HLA-DR on peripheral blood monocytes,the compositions of the present invention further comprise amonocyte-distinguishing antibody, preferably an anti-CD14 antibody, morepreferably an anti-CD14 antibody conjugated to a fluorescence energytransfer tandem fluorophore, most preferably conjugated to the PerCPmoiety of PerCP/CY5.5.

The present methods are particularly useful in clinical settings andwill thus likely be performed, inter alia, by clinical laboratories.However, any one clinical laboratory may have only sporadic need toperform such methods. The invention thus provides, in another aspect,kits that permit the assay readily to be performed on an as-neededbasis.

In preferred embodiments, a kit of the present invention comprises oneor more staining compositions and an erythrocyte-lysing composition. Atleast one staining composition comprises an anti-HLA-DR antibody and alysosomotropic amine, preferably chloroquine. In preferred embodiments,the anti-HLA-DR antibody is conjugated to PE, preferably at a definedmolar ratio, most preferably at a ratio of 1:1. In embodiments usefulfor quantitating HLA-DR on the surface of peripheral blood monocytes, atleast one staining composition of the kit comprises amonocyte-distinguishing antibody, preferably anti-CD14. In preferredembodiments, the anti-CD14 antibody is conjugated to a fluorescenceresonance transfer tandem fluorophore, most preferably PerCP/CY5.5. Inparticularly preferred embodiments, the kit contains a stainingcomposition that comprises an anti-HLA-DR-PE antibody, ananti-CD14-PerCP/CY5.5 antibody, and chloroquine.

In other embodiments, the kit comprises one or more stainingcompositions, an erythrocyte lysing composition, and further comprisespelletized beads conjugated with defined levels of PE. In theseembodiments, the anti-HLA-DR antibody is conjugated to PE and thepelletized beads permit the calibration of the flow cytometer to allowquantitation, from the measured PE fluorescence, of the amount of HLA-DRantibody bound per cell.

In another aspect, the invention provides a novel monocyte-specificimmunoconjugate, comprising an anti-CD14 antibody conjugated to thePerCP moiety of a PerCP/CY5.5 tandem dye molecule. The specificity ofthe CY5.5 moiety for CD64, coupled with the specificity of the antibodyfor CD14, provides a high intensity monocyte-specific signal for singlelaser, multicolor fluorescence activated cell sorting applications.

In yet another aspect, the invention provides an improved method ofmeasuring CD11b expression on the surface of human blood cells,comprising contacting a sample containing human blood cells with alysosomotropic amine and an antibody specific for CD11b; and thendetecting the binding of the CD11b antibody to the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages of the present invention willbe apparent upon consideration of the following detailed descriptiontaken in conjunction with the accompanying drawings, in which:

FIG. 1 is a graph demonstrating the time-dependence of HLA-DR-specificfluorescent signal reported by CD14⁺ monocytes incubated for increasingtimes with an anti-HLA-DR antibody;

FIG. 2 presents graphs demonstrating the ability of chloroquine—but notof azide, Brefeldin A or monensin—to stabilize the HLA-DR-specificfluorescent signal measured flow cytometrically from monocytes ofdifferent individuals, with FIGS. 2D and 2E showing the results ofchloroquine titration experiments;

FIG. 3 shows dot plots of peripheral blood cells from two individuals,demonstrating wide range of CD14-specific FITC fluorescence in the CD86⁺population;

FIG. 4 shows dot plots of peripheral blood cells drawn from the same twoindividuals as in FIG. 3, demonstrating strong clustering of CD14⁺monocytes (circled) using an anti-CD14 antibody conjugated to the PerCPmoiety of a PerCP/CY5.5 tandem fluorophore;

FIG. 5 shows data obtained from a sample of patient peripheral bloodusing the reagents and methods of the present invention;

FIG. 6 plots monocytic HLA-DR expression data obtained using thereagents and methods of the present invention against data from the samesamples obtained using a prior approach; and

FIG. 7 demonstrates stabilization by chloroquine of CD11b-specificfluorescent signal measured flow cytometrically in a whole blood sample.

DETAILED DESCRIPTION OF THE INVENTION

In the description, the following term is used. “Monocyte-distinguishingantibody” refers to any antibody that may be used alone, in combinationwith a cell's physical properties, such as light-scattering properties,or in combination with one or more additional antibodies, to distinguishmonocytes in a peripheral blood sample. The term thus includes, interalia, an anti-CD14 antibody.

Recent clinical studies suggest that proinflammatory cytokines, such asIFN-γ and G-CSF, may be used successfully to treat those septic patientsin whom the early, proinflammatory response has given way to a systemicimmunosuppression, also termed an “immune paralysis”. Further evidencesuggests that persistent reduction in HLA-DR expression on peripheralblood monocytes is a useful clinical marker of this immunosuppressedstate, and that therapeutic decisions may successfully be based uponsuch measurements.

Although reagents and methods presently exist that permit themeasurement of HLA-DR on the surface of monocytes, two problems readilyattend attempts to develop an improved flow cytometric assay that is atonce rapid, reproducible, and quantitative.

The first relates to the dynamic nature of HLA-DR expression on thesurface of professional APCs such as monocytes. As further describedbelow in Example 1, peripheral blood samples from normal donors wereincubated with a commercially-available PE-conjugated anti-HLA-DRantibody. Also present in the incubation was a monocyte-distinguishingantibody, anti-CD14. FIG. 1 demonstrates that the incubation ofperipheral blood samples with an anti-HLA-DR antibody gives rise to atime-dependent increase in HLA-DR-specific fluorescence measured flowcytometrically on the CD14⁺ monocytes. Furthermore, the slope of theincrease depends upon the individual donor; as a result, it cannotreadily be controlled by a simple and universal mathematical adjustment.

The basis for this time-dependence of HLA-DR fluorescence signal isuncertain.

Part of the mechanistic uncertainty no doubt derives from the ability ofthe flow cytometer readily to detect fluorescence from internalized, aswell as cell surface-bound, antibody. Picker et al., Blood86(4):1408-1419 (1995); Suni et al., J. Immunol. 212:89-98 (1998). MHCclass II antigens are known to be internalized and then recycled. Theprocess is rapid, with a half-time for endocytosis of the surface classII population of 33 minutes; recycling traffic may in fact exceed HLA-DRbiosynthetic traffic by some 60-fold. Reid et al., Nature 346:655-57(1990). While not wishing to be bound by theory, it is possible that theincrease in HLA-DR signal that is observed is occasioned by theprogressive internal accumulation of protein labeled during its passageon the cell surface.

Uncertainty also derives from the very complexity of the traffickingroutes. Class II MHC glycoproteins traverse a number of intracellularcompartments during biosynthetic maturation, Cresswell, Annu. Rev.Immunol. 12:259-93 (1994), and recycling occurs through disparate, butlikely intersecting, pathways. Watts, Annu. Rev. Immunol. 15:821-50(1997).

The present invention is based in part upon the surprising finding thatchloroquine, when included in the incubation with anti-HLA antibody,stabilizes the HLA-DR-specific signal obtained from CD14⁺ monocytes.This finding is particularly surprising given chloroquine's reportedinability to block the recycling of MHC glycoproteins, Reid et al.,Nature 346:655-57 (1990).

FIG. 2 demonstrates that addition of chloroquine at a finalconcentration of 3 mM stabilizes the HLA-DR fluorescence signal. Azide(FIG. 2A), Brefeldin A (FIG. 2C) and monensin (FIG. 2C), the latter twoof which are known to interfere with Golgi-related protein processingsteps, provide no significant stabilization.

Thus, inclusion of chloroquine during the incubation with anti-HLA-DRantibody renders the result substantially independent of the duration ofincubation, from approximately 15 minutes up to the maximum testedperiod of 60 minutes. This substantially increases the reliability,reproducibility, and utility of this assay for clinical use.

Although the incubation shown in FIG. 2 ranges from about 15 minutes toabout 60 minutes, incubation is preferably 20-50 minutes, morepreferably 25-45 minutes, most preferably 25-35 minutes. And althoughchloroquine is exemplified and presently preferred, other lysosomotropicamines that stabilize the HLA-DR-specific fluorescent signal may also beused. Lysosomotropic amines include, e.g., chloroquine,hydroxychloroquine, primaquine, and methylamine.

FIGS. 2D and 2E present chloroquine titration experiments, demonstratingthat a minimum concentration of 2 mM chloroquine is needed to effectHLA-DR stabilization in the assay. Chloroquine is thus included in theincubation at a final concentration of 2.0-50 mM, preferably 2-5 mM,more preferably at a final concentration of 2-3 mM, most preferably at afinal concentration of about 3 mM. Determination of concentration ofchloroquine or other lysosomotropic amine that is best suited to thestable measurement of HLA-DR is readily determined using standardtitration experiments, such as are reported in Example 2, below.

In preferred embodiments of the method and reagents of the presentinvention, the anti-HLA-DR antibody is specific for nonpolymorphic classII determinants, and may thus be used for measurement of HLA-DR inmultiple individuals, preferably all human individuals. It is alsopreferred that the antibody not cross-react with HLA-DQ or HLA-DPmolecules. The experiments reported herein used anti-HLA-DR antibodiesfrom clone L243.

In addition, although the methods of the present invention may use avariety of anti-HLA-DR antibodies conjugated to a variety offluorophores, a preferred embodiment of the methods of the presentinvention utilizes HLA-DR antibodies that permit the quantitation ofHLA-DR surface density.

A number of recent reports demonstrate the advantages of measuring theamount, or density, of antigen expression on cell populations, ascompared with simply assaying the frequency of cells expressing theantigen. Poncelet et al., Eur. J. Histochem. 1:15-32 (1996);Lavabre-Bertrand, Eur. J. Histochem. 1:33-38 (1996); Liu et al.,Cytometry 26:1-7 (1996); Moore, Science 276:51-52 (1997); Rehse et al.,Cytometry 22: 317-322 (1995); Patel et al., “Anal. Biochem. 229:229-235(1995). Quantitative methodology is especially important incharacterizing cell populations that express heterogeneous levels ofantigen and in characterizing expression of antigens whose levels changedynamically. Monocyte expression of HLA-DR meets both these criteria.

In the context of immunostimulatory treatment of sepsis, finerdiscrimination of monocytic HLA-DR surface levels should lead to moreaccurate and improved discrimination of the patient populations likelyto benefit from therapy, avoiding immunostimulation of clinicallyinappropriate patients. As to those patients selected for treatment withIFN-γ, G-CSF, or other immunostimulants, finer discrimination ofmonocytic HLA-DR levels should permit dosage individualization basedupon the density distribution of HLA-DR on each patient's peripheralblood monocytes; in the reported pilot study, all patients meeting theinclusion criteria were given a uniform dose of interferon.

Yet a further advantage of quantitative analysis is that suchquantitation renders the assay essentially independent of laboratory tolaboratory variation. In contrast, qualitative classification, such asdefining positively-staining populations as bright or dim, is bothsubjective and highly dependent upon the measuring instrument and itssettings. For example, in the clinical trial reported by both Kox etal., Arch. Intern. Med. 157:389-393 (1997) and Döcke et al., Nature Med.3:678-681 (1997), the PE fluorescence intensity cutoff used todistinguish the HLA-DR⁻ from HLA-DR⁺ populations was both idiosyncraticand machine-dependent. Additionally, the use of such a criterion todefine two populations, one positive, one negative, provides only acrude measure of the distribution of HLA-DR densities in the monocytepopulation.

Thus, a preferred embodiment of the compositions, methods and kits ofthe present invention utilize HLA-DR antibodies that permit thequantitation of HLA-DR surface density. Preferably, such antibodies areconjugated to phycoerythrin (PE), more preferably at a defined PE:Abmolar ratio, most preferably at a 1:1 molar ratio.

PE is particularly well-suited to such quantitative analysis: it isbright, and it does not self-quench. Fluorophores such as fluoresceinisothiocyanate (FITC), CY3 and CY5 can self-quench due to overlap oftheir excitation and emission spectra. Quenching of FITC has been shownto occur both through proximity of FITC molecules on a single antibodyand through the proximity of FITC molecules on adjacent antibodiesdirected to a high density cellular epitope. Haugland, Handbook ofFluorescent Probes and Research Chemicals, 6th ed., Molecular ProbesInc., Eugene, Oreg. (1996); Deka et al., Cytometry 25:271-279 (1996).Peridinium chlorophyll protein (PerCP), which is described, inter alia,in U.S. Pat. No. 4,876,190, incorporated herein by reference, photolysesduring its transit through the laser of the flow cytometer, precludingaccurate quantitation. Other advantages of PE for antigen densityquantitation are described in U.S. Pat. No. 5,620,842, incorporatedherein by reference.

A further advantage of PE as a fluorophore is the commercialavailability of anti-HLA-DR antibodies conjugated at defined molarratios of PE (Becton Dickinson Immunocytometry Systems, San Jose,Calif.) and the commercial availability of pelletized bead standards(QuantiBRITE™, BDIS), the beads providing defined levels of PEfluorescence.

The second problem encountered in attempts to develop an improved flowcytometric assay for HLA-DR expression on peripheral blood monocytes isthat anti-CD14 antibodies labeled with FITC and with other common,PE-distinguishable, fluorophores do not permit the unambiguousdiscrimination of monocytes from granulocytes. A sufficient percentageof monocytes stain dimly with anti-CD14 as to overlap the weakly-stainedgranulocytic population. This is shown in FIG. 3, dot plots from twoseparate patients demonstrating, in each case, the wide range ofanti-CD14-FITC fluorescence in the CD86⁺ population in peripheral blood.

The present invention is based, in part, upon the novel finding thatconjugating an anti-CD14 antibody to the PerCP moiety of a PerCP/Cy5.5resonance energy transfer tandem fluorophore provides a highly specific,highly uniform, high intensity clustered signal from peripheral bloodmonocytes, permitting the ready discrimination of monocytes fromgranulocytes.

Cyanine resonance energy transfer tandem fluorophores (“tandemfluorophores”, “tandem dyes”, “tricolor stains”) have recently expandedthe choices of fluorophore available for single-laser, multi-color flowcytometric analysis. PE-CY5 tandem staining proves particularlywell-suited for three-color analysis: the R-PE moiety, excited by the488 nm light of an argon ion laser, serves as an energy donor, and CY5,acting as an energy acceptor, fluoresces at 670 nm, readilydistinguishable from the emission of FITC and PE. Cyanine fluorophoresare described in U.S. Pat Nos. 5,268,486; 4,337,063; 4,404,289;4,405,711; and in Mujumdar et al., Bioconj. Chem. 4:105-111 (1993);Southwick et al., Cytometry 11:418-430 (1990); Ernst et al., Cytometry10:3-10 (1989); and Mujumdar et al., Cytometry 10:11-19 (1989), andcyanine energy resonance transfer tandem fluorophores are described,inter alia, in U.S. Pat. No. 5,714,386 and in Waggoner et al., Ann. NYAcad. Sci. 677:185-193 (1993) and Lansdorp et al., Cytometry 12:723-30(1991), the disclosures of which are incorporated herein by reference.

However, the CY5 moiety of these tandem fluorophores has been shown tobind directly to CD64 (FcγRI) giving spurious, antibody-independent,signals on CD64⁺ cells. van Vugt et al., Blood 88:2358-2360 (1996). Thisantibody-independent binding has motivated considerable efforts in theart to mask the CY5 moiety to reduce the spurious binding to CD64molecules presented on the surface of peripheral blood cells asdescribed, inter alia, in U.S. patent application Ser. No. 08/943,491,filed Oct. 3, 1997, which issued as U.S. Pat. No. 6,133,429, thedisclosure of which is incorporated herein by reference in its entirety.

But CD64 is constitutively expressed on monocytes and macrophages, andis strongly up-regulated by IFN-γ, making it a usefulmonocyte-distinguishing marker.

Reversing prior art efforts to bring the CY5 moiety into increasingproximity to the antibody to mask its CD64 specificity, a novelanti-CD14 conjugate was prepared in which the CY5.5 moiety of a tandemPerCP/CY5.5 fluorophore was purposely oriented to facilitate itsinteraction with CD64. In this conjugate, the anti-CD14 antibody wasconjugated to the PerCP moiety of a PerCP/CY5.5 tandem dye, thusdisplaying the CY5.5 moiety at some distance from the antibody.

FIG. 4 shows the strong clustering of CD14⁺ monocytes observed usingthis novel CD14-PerCP/CY5.5 conjugate. The samples are drawn from thesame patients as those for whom data is shown in FIG. 3. AlthoughPerCP/CY5.5 is exemplified, the tandem fluorophore PE/CY5 may also beused. PerCP/CY5 has been found ineffective in the present methods.

In one aspect, then, the instant invention provides a method ofmeasuring HLA-DR surface expression on human peripheral blood monocytesthat incorporates both of these improvements. In preferred embodiments,a peripheral blood sample is contacted in the presence of chloroquinewith a first antibody, the first antibody specific for HLA-DR andconjugated to PE, and also with a second antibody, the second antibodyspecific for CD14 and conjugated to the PerCP moiety of a PerCP/CY5.5tandem fluorophore; the binding of the anti-HLA-DR antibody to the CD14⁺cells is then measured, preferably by flow cytometry.

The antibody incubation is preferably performed on anunfractionated—that is, whole blood peripheral—blood sample. Althoughthe methods, reagents, and kits of the present invention may equally beused to assess HLA-DR expression on monocyte-containing peripheral bloodfractions, the elimination of antecedent fractionation steps effects asimplification over the protocol used in the reported clinical trial,Kox et al., Arch. Intern. Med. 157:389-393 (1997); Döcke et al., NatureMed. 3:678-681 (1997); Docke et al., in Schmitz et al. (eds.),Durchflusszytometrie in der Klinischen Zelldiagnostic, Schattauer:Stuttgart and New York (1994), pp. 163-177 (hereinafter collectively“Döcke assay”). Eliminating fractionation steps reduces assay time,reduces potential sample loss, and reduces sources of assay variance,important benefits in an assay intended, in part, for clinical use.Further advantages of whole blood assays are reported in Picker et al.,Blood 86(4):1408-1419 (1995); Suni et al., J. Immunol. 212:89-98 (1998);and Kahan, “Application Note 2: Detecting Intracellular Cytokines inActivated Monocytes,” Becton Dickinson Immunocytometry Systems (SanJose) (1997), the disclosures of which are incorporated herein byreference.

As would be apparent, however, the persistence of erythrocytes in thewhole blood sample beyond the staining step may complicate later effortsto measure fluorophore bound specifically to the nucleated cells in thesample. Thus, in such circumstances the methods of the present inventionmay include, after the antibody incubation step and before the flowcytometric acquisition of data, a further step of lysing theerythrocytes in the sample.

A number of agents that serve simultaneously to lyse red blood cells andto fix nucleated cells in a whole blood sample, without interfering withbinding of antibody to the nucleated cells, are known in the art. Theseagents are described, inter alia, in U.S. Pat. Nos. 4,654,312;4,902,613; 5,510,267; 5,516,695; 5,648,225 and European Patent No. EP161770 B1, the disclosures of which are incorporated herein byreference. Several such agents are available commercially, includingFACS™ Lysing Solution (Becton Dickinson Immunocytometry Systems, SanJose, Calif., catalogue No. 349202) and Whole Blood Lysing Solution(Caltag Laboratories, Inc., Burlingame, Calif., catalogue no. GAS-10).

The minimum duration of incubation with lysis reagent depends uponwhether, after lysis, debris is removed by centrifugation.

In the simplest assay protocol, flow cytometric determination of HLA-DRexpression is measured directly from the lysed sample, without removalof lysis debris. In this approach, the incubation with lysis reagent mayrange from about 1-60 minutes, preferably from about 2-30 minutes, morepreferably about 10 minutes.

In an alternative protocol in which lysis debris is removed bycentrifugation, followed optionally by washing of the mononuclear cellpellet, incubation with lysis reagent is preferably performed for aminimum of about 8-10 minutes prior to centrifugation. In thisalternative protocol, incubation ranges from about 8-60 minutes,preferably about 8-30 minutes, most preferably from about 8-15 minutes,with an incubation of about 8-10 minutes most preferable.

Incubation with lysis reagent in either case is performed preferably atroom temperature, in accordance with the instructions packaged with thelysis reagent.

Thus, as set forth in Example 1 below, a preferred protocol formeasuring HLA-DR expression on the surface of peripheral blood monocytesis as follows:

-   1) To 50 μl of whole blood add 10 μl of staining reagent. The    staining reagent contains

500 ng of HLA-DR-PE (1:1 Ab:PE ratio)

63 ng CD14-PerCP/CY5.5

18 mM chloroquine

-   in buffered saline containing gelatin and 0.1% sodium azide (where,    in accordance with convention, HLA-DR-PE and CD14-PerCP/Cy5.5 refer    to antibodies with specificity for HLA-DR and CD14, respectively, as    conjugated to the indicated fluorophores)-   2) Incubate for 25-35 minutes in the dark at room temperature.-   3) Add 1 ml 1× FACS™ Lysing Solution (obtained as 10× stock, BDIS    catalog No. 349202) and incubate approximately 10 minutes at room    temperature in the dark.-   4) Analyze directly using a FACS™ brand flow cytometer (BDIS, San    Jose, Calif.). Acquire 2000 monocyte events and assess the binding    of anti-HLA-DR antibody thereto.

As would be apparent to the skilled artisan, the method for calibratingand running the flow cytometer, as well as the methods for analyzing thedata so acquired, can be varied, and such variations are well within theskill in the art.

And as would be further understood, flow cytometers and fluorescenceactivated cell sorters from other manufacturers can also be used, as canother devices that prove useful in measuring fluorescence fromantibody-labeled cells, such as microvolume fluorimeters (volumetriccapillary cytometers), such as are described in U.S. Pat. Nos.5,547,849, 5,556,764, 5,932,428 and 5,912,134, incorporated herein byreference in their entireties.

And as further described in the Examples below, further steps may beperformed, such as removal of lysis debris by aspiration of supernatantfollowing collection of the cells by centrifugation, optionallyincluding a further wash of the mononuclear cell pellet.

It will also be apparent that monocytes may, and will preferably, bedistinguished not only by the CD14⁺ fluorescent signal but also byphysical properties reported, e.g., by the degree of side scatter andforward scatter. Monocytes may even be distinguished solely on the basisof such physical properties.

Finally, it should be noted that samples should not be left too longdiluted at the final step prior to data acquisition; about 6% of thesignal is lost in the first hour.

Where the anti-HLA-DR antibody is conjugated to fluorophore at a definedmolar ratio, it is possible in the above-described assay to estimate thedensity distribution of HLA-DR expression on the CD14⁺ monocytepopulation, and to report the same as antibodies bound per cell.

As set forth in Example 2, peripheral blood samples were obtained frompatients and the density of HLA-DR assayed on monocytes usingchloroquine incubation and an anti-CD14-PerCP/CY5.5 conjugate. Asfurther described in the Example, the protocol, in contrast to thatpresented above, included removal of lysis debris by centrifugation.Samples were also assayed according to the prior Döcke method. Resultsare shown in FIGS. 5 and 6.

FIG. 5A plots sidescatter (SSC) versus fluorescence channel 3(CD14-PerCP/CY5.5) for two aliquots of a single patient sample processedaccording to the method of the present invention. The tightly-clusteredCD14⁺ monocyte population is circled on each. FIG. 5B shows, directlybelow each dot plot, a histogram charting the PE fluorescence intensity(HLA-DR) measured from the cells in the demarcated monocyte population.Histogram statistics are also given.

FIG. 6 compares the results obtained using methods of the presentinvention with results obtained using the prior Döcke assay. The y-axisreports number of phycoerythrin molecules (anti-HLA-DR PE) bound onaverage per monocyte using the present methods; the x-axis reports thepercentage of monocytes positive for HLA-DR using the prior methods.Correlation is high, with a correlation coefficient (r²) of 0.908.

To obtain reliable percentages of HLA-DR⁺ monocytes according to theDöcke method, it was necessary to stain cells using subsaturation levels(approximately 20% saturation) of anti-HLA-DR PE antibody and to monitorthe incubation time carefully, both per published protocol. The lowdegree of antibody saturation, however, means that small variations inantibody concentration lead to large changes in antibody binding, andthus of measured fluorescence. The care with which antibody must betitrated and the degree to which incubation time must be disciplinedremove the Döcke assay from the realm of the clinical routineer.

The high correlation between the percentage HLA-DR⁺ monocytes asmeasured by the Döcke method and the number of anti-HLA-DR PE moleculesbound per monocyte as measured by the methods of the present inventionpermits the direct translation of the established clinical criteria tomeasurements reported by the present assay. Thus, as seen in FIG. 6, theprior-established treatment criterion of 30% HLA-DR⁺ monocytestranslates to approximately 3000 molecules PE (anti-HLA-DR) bound permonocyte; similarly, the prior criterion for cessation of treatment, 50%HLA-DR⁺ monocytes, translates to approximately 4,750 molecules of PE(anti-HLA-DR) bound per cell.

Thus, in another aspect, the present invention provides a method ofdetermining the suitability of immunostimulatory therapy in a patientwith sepsis. The method comprises, in its most preferred embodiment, thesteps of contacting a peripheral blood sample from the patient with ananti-HLA-DR PE antibody and an anti-CD14-PerCP/CY 5.5 antibody in thepresence of chloroquine, then measuring the number of HLA-DR antibodiesbound per CD14⁺ monocyte. Using the prior-established clinical treatmentparameters, patients averaging fewer than 3000 anti-HLA-DR antibodiesper monocyte for two consecutive days are determined to be suitable forcommencement of immunostimulatory treatment, such as treatment withIFN-γ, and treatment is continued until that average density rises toapproximately 4700-5000, preferably 5000 or more, anti-HLA-DR moleculesper peripheral blood monocyte, preferably for two to three consecutivedays.

It will, of course, be understood that with the accumulation of moreclinical data, which data will now increasingly be forthcoming given theease and reliability of the methods of the present invention, thatcriteria for immunostimulatory treatment of septic patients likely willchange. Thus, it should be understood that the present inventionprovides a general method of determining the suitability ofimmunostimulatory therapy in a patient with sepsis, wherein the densityof HLA-DR expression on the surface of peripheral blood monocytesindicates a measure of immune dysfunction in the patient's professionalAPC compartment, suitable for remediation by immunostimulatory agentsand methods. It will also be understood that IFN-γ, while the presentlypreferred immunostimulatory agent, will likely be complemented in thefuture or even superseded by other agents that reverse the dysfunctionof the patient's professional APCs. Among such methods are treatmentwith G-CSF, GM-CSF and dialysis.

As noted, the methods presented herein are of particular utility in theclinical setting. Flow cytometry has now become a routine part of theclinical laboratory, Riley et al., Clinical Applications of FlowCytometry, Igaku-Shoin Medical Publ. (1993); Coon et al. (eds.),Diagnostic Flow Cytometry (Techniques in Diagnostic Pathology, No 2),Williams & Wilkins (1991); Keren et al., Flow Cytometry and ClinicalDiagnosis, Amer. Soc'y of Clinical Pathol.(ISBN 0891893466, 1994), andthe methods presented herein are well within the skill of the clinicalpathology laboratory.

However, any one clinical laboratory may have only sporadic need toperform the assay, and there is thus a need for compositions and kitsthat permit the assay readily to be performed on an as-needed basis.

Thus, in another aspect, the present invention provides compositions,hereinafter termed “staining compositions,” each comprisingfluorophore-conjugated antibody and a lysosomotropic amine. In oneembodiment, the staining composition comprises an anti-HLA-DR antibodyand a lysosomotropic amine. More preferably, the staining compositionfurther comprises an anti-CD14 antibody; in the most preferredembodiments, the composition comprises anti-HLA-DR-PE,anti-CD14-PerCP/CY5.5, and chloroquine. For measurement of the densityof HLA-DR molecules on the surface of monocytes, the anti-HLA-DRantibody is conjugated to PE at a defined molar ratio, most preferablyat a molar ratio of 1:1.

It will be understood that these staining compositions may furtherinclude various diluents, preservatives and stabilizers standard in theart, such as buffered gelatin and sodium azide.

In yet another aspect, the present invention provides kits for themeasurement of HLA-DR on the surface of peripheral blood cells, and inpreferred embodiments, on the surface of peripheral blood monocytes.

In one embodiment, the kit comprises a staining composition and anerythrocyte lysing composition. The staining composition, as describedabove, preferably comprises an anti-HLA-DR antibody and a lysosomotropicamine. More preferably, the staining composition further comprises ananti-CD14 antibody; in the most preferred embodiments, the compositioncomprises anti-HLA-DR-PE, anti-CD14-PerCP/CY5.5, and chloroquine. Forapplications in which the density of HLA-DR is to be measured, theanti-HLA antibody of the staining composition of the kit is conjugatedto PE at a defined molar ratio, most preferably at a molar ratio of 1:1,and the kit further comprises pelletized beads conjugated with definedlevels of PE, to permit the calibration of the flow cytometer.

As noted above, the basis for the time-dependence of the HLA-DRfluorescence signal during anti-HLA-DR antibody staining is unknown, asis the mechanism by which chloroquine stabilizes the signal over time.But whatever the mechanism, it is clear that it is shared by other cellsurface molecules. Thus, FIG. 7 shows that the fluorescence signalproduced during antibody staining of CD11b in whole blood samples alsoincreases with staining time, and that the fluorescent signal cansimilarly be stabilized by addition of chloroquine at 3 mM finalconcentration. FIG. 7 further demonstrates that these phenomena, as withHLA-DR, can be seen in the blood from donors with quite differentaverage levels of expression.

Thus, in another aspect, the invention provides improved methods,compositions, and kits for measuring CD11b expression on the surface ofhuman blood cells. The method comprises contacting a sample containinghuman blood cells with a lysosomotropic amine and an antibody specificfor CD11b, and then detecting the binding of the CD11b antibody to thecells. The compositions comprise a CD11b-specific antibody, typicallyconjugated to a fluorophore, and a lysosomotropic amine, typicallychloroquine. In one embodiment, the kit comprises a staining compositionand an erythrocyte lysing composition. The staining composition, asdescribed above, preferably comprises an anti-CD11b antibody and alysosomotropic amine. The staining composition can further comprise ananti-CD45 antibody; in the most preferred embodiments, the compositioncomprises anti-CD11b-PE, anti-CD45 PerCP, and chloroquine.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

In general, and unless otherwise stated, the flow cytometric methodsused in the following examples are well known in the art. Detailedprotocols are compiled in several recent compendia, including FlowCytometry: A Practical Approach, 2nd ed., M. G. Ormerod (ed.), OxfordUniversity Press, 1997; Handbook of Flow Cytometry Methods, J. PaulRobinson (ed.), John Wiley & Sons (1993); Current Protocols inCytometry, J. Paul Robinson (ed.), John Wiley & Sons (October 1997, withperiodic updates); Becton Dickinson Cytometry Source Book, BectonDickinson Immunocytometry Systems (1998, with periodic updates)(SanJose, Calif.), the disclosures of which are herein incorporated byreference.

For convenience, antibody designations are abbreviated by indicatingantigenic specificity followed by fluorophore. Fluorophore abbreviationsare phycoerythrin (PE), peridinin chlorophyll protein (PerCP),allophycocyanin (APC), fluorescein isothiocyanate (FITC), cyanine 5(CY5) and cyanine 5.5 (CY5.5). Thus, an antibody labeled withphycoerythrin (PE) that is specific for HLA-DR is denominated “HLA-DRPE.” Becton Dickinson Immunocytometry Systems (San Jose, Calif.) as thesource of reagents is abbreviated “BDIS”.

Example 1 Chloroquine Inhibition of Time-Dependent Variation inHLA-DR-Specific Fluorescent Antibody Staining

Peripheral blood samples were drawn from normal volunteers.

The time-dependence of the HLA-DR-specific signal detectable by flowcytometry of antibody-stained peripheral blood monocytes was assessed byvarying the duration of antibody staining.

For the experiments reported in FIG. 1, to 50 μl EDTA-anticoagulatedwhole blood was added 10 μl of staining reagent containing either (a)CD14PerCP/CY5.5 (63 ng) and HLA-DR-PE (250 ng) (donors #160 and #325) or(b) CD14-PerCP/CY5.5 (63 ng) and HLA-DR-PE (500 ng) (donor #790).

Samples were incubated with staining reagent at room temperature for thetimes indicated, after which 1 ml of 1× FACS™ Lysing Solution (10× stockfrom BDIS, catalog no. 349202) was added, and incubation continued for10 minutes.

For donor #160, the samples were then centrifuged at 250×g for 5minutes, the supernatant removed, the pellet resuspended in 2 ml ofPBS+0.5% BSA and recentrifuged. The final pellet was suspended in 0.5 mlof PBS+BSA and analyzed by flow cytometry. For donors #790 and #325 thepost-lysis wash was omitted: following the incubation with LysingSolution, the samples were centrifuged at 250×g for 5 minutes, thesupernatant removed, and the pellet directly resuspended in 0.5 ml ofPBS+BSA for analysis by flow cytometry.

Flow cytometric acquisition of at least 2000 monocyte events wasperformed for each sample using a FACS™ brand flow cytometer (BDIS)calibrated using CaliBRITE™ (BDIS cat. no. 349502) or QuantiBRITE™ (BDIScat. no. 340495) beads according to manufacturer's instructions. The PEfluorescence intensity for monocyte-specific events was plotted as shownin FIG. 1.

The effect of various additives on the stability of HLA-DR-specificfluorescence intensity was tested by including these reagents during thestaining step.

In a first experiment, the results of which are reported in FIG. 2A,peripheral blood samples (50 μl EDTA-anticoagulated whole blood) werestained with either (a) 10 μl of staining reagent containing 500 ngHLA-DR-PE and 63 ng CD14-PerCP/CY5.5 (reported in the figure as“Control”); (b) 10 μl staining reagent containing 500 ng HLA-DR-PE, 63ng CD14-PerCP/CY5.5, and 0.6% sodium azide (final azide concentration0.1%; reported in FIG. 2A as “Azide”); or (c) 10 μl staining reagentcontaining 500 ng HLA-DR-PE, 63 ng CD14-PerCP/CY5.5, and 18 mMchloroquine (final chloroquine concentration 3 mM; reported in FIG. 2Aas “Chloroquine”). Incubation was at room temperature for the timesindicated, after which 1 ml of 1× FACS™ Lysing Solution was added andincubation continued for 10 minutes.

Following erythrocyte lysis, the samples were centrifuged at 250×g for 5minutes, the supernatant removed, the pellet resuspended in 2 ml ofPBS+0.5% BSA and recentrifuged. The final pellet was suspended in 0.5 mlof PBS+BSA and analyzed by flow cytometry, as above. Results are plottedin FIG. 2A.

A second experiment, reported in FIG. 2B, was performed similarly, butomitting the post-lysis wash step: following erythrocyte lysis, thesamples were centrifuged at 250×g for 5 minutes, the supernatant,containing lysis debris, removed, and the pellet resuspended directly in0.5 ml of PBS+0.5% BSA. Again, flow cytometric acquisition of at least2000 monocyte events was performed, and the PE fluorescence intensity ofthe monocyte population plotted.

To test whether Brefeldin A and monensin would similarly stabilizeHLA-DR expression in the flow cytometric assay, an additional series ofexperiments was performed.

To 50 μl EDTA-anticoagulated whole blood was added 10 μl stainingreagent (500 ng of HLA-DR-PE and 63 ng CD14-PerCP/CY5.5 in bufferedsaline containing gelatin and 0.1% sodium azide); the staining reagentfurther included the additives noted below. Samples were incubated withstaining reagent for the times indicated, after which 1 ml of 1× FACS™Lysing Solution was added. After incubation with lysing solution for 9.5to 11.5 minutes, samples were directly analyzed using a FACSCalibur™brand flow cytometer (BDIS, San Jose, Calif.).

Additives to the staining composition for this latter series ofexperiments, reported in FIG. 2C, were: (A) nothing (“Control”); (B) 18mM chloroquine; (C) 0.2 mM Brefeldin A; and (D) 0.013 mM monensin. Finalconcentrations of additives after 1:6 dilution during staining were: (B)3 mM chloroquine; (C) 0.034 mM Brefeldin A; and (D) 0.0022mM monensin.Final concentrations of Brefeldin A and monensin are those that havebeen found sufficient to block processing and secretion of cytokines,per Picker et al., Blood 86(4):1408-1419 (1995); Suni et al., J.Immunol. 212:89-98 (1998).

The data presented in FIG. 2C were acquired immediately after lysis; incontrast to experiments the results of which are presented in FIG. 1,the removal of lysis debris (by centrifugation and aspiration,optionally followed by wash) was omitted. Independent experiments (notshown) have demonstrated that the results are comparable whether debrisis removed or not. When debris removal is not performed, the duration ofthe lysis step may be shortened: samples can be analyzed as soon asapproximately 60 seconds after staining. Samples may be assayed up toabout 60 minutes after staining (with loss, over the 60 minutes, ofabout 5-10% HLA-DR-PE fluorescence).

As shown in FIGS. 2A-2C, chloroquine—but not azide, Brefeldin A ormonensin—reliably stabilized HLA-DR fluorescence, with signals at 60minute incubation substantially identical to those at 10-15 minutesincubation. In experiments not shown, hydroxychloroquine was shownequally effective in stabilizing HLA-DR signal as chloroquine.

A further experiment was performed to determine the minimumconcentration of chloroquine needed to effect stabilization of HLA-DRexpression on the surface of monocytes.

An aliquot of 50 μl peripheral blood was drawn from a healthy volunteerand mixed with 10 μl HLA-DR quantitation reagent (50 μg/ml HLA-DR PE,6.3 μg/ml CD14 PerCP-Cy5.5, in 10 mM sodium pyrophosphate, 0.15 M NaCl,pH 8). The HLA-DR quantitation reagent further contained chloroquine at0 mM (control), 6 mM, 12 mM, or 18 mM.

In each experiment, 4 blood samples were stained for each chloroquinelevel, with one each of the samples stained for 20, 40, 60 or 80minutes.

At the end of the staining period, the samples were lysed with 1× FACS™Lysing Solution (BDIS) and analyzed on a FACScalibur Flow Cytometer.Five hundred monocyte events were collected. The HLA-DR PE median (FL2)was determined from a histogram which was gated on the monocytepopulation in FL3\side scatter. Results, shown in FIGS. 2D and 2E,demonstrate that HLA-DR stabilization may be obtained with chloroquineat 2 mM or above, but that 1 mM chloroquine was insufficient to effectstabilization.

Example 2 Measurement of HLA-DR on Patient Peripheral Blood Monocytes

Peripheral blood samples were obtained by venipuncture from patientsadmitted to ICU. One aliquot of each sample was assayed using thefollowing protocol.

To 50 μl of whole blood was added 10 μl of staining reagent containing50g/ml HLA-DR-PE (clone L243); 6.3 μg/ml CD14-PerCP/CY5.5; and 18 mMchloroquine diphosphate. HLA-DR-PE (BDIS cat. no. 347367, repurified toprovide a 1:1 antibody:PE conjugate) is a mouse IgG_(2a)/κ antibody thatreacts with a nonpolymorphic HLA-DR epitope and does not cross-reactwith HLA-DQ or HLA-DP molecules. CD14-PerCP/CY5.5 (BDIS custom conjugateof antibody LeuM3, available unconjugated as BDIS catalogue number347490) is a mouse IgG_(2b)/K antibody specific for CD14.

The aliquot was incubated for 25-35 minutes in the dark at roomtemperature, and then 1 ml 1× FACS™ Lysing Solution (obtained as 10×stock, BDIS catalog No. 349202) was added and incubation continued for8-10 minutes. The cells were then collected by centrifugation at 300×g(1200 RPM in a Sorvall 6000 centrifuge) and the lysis debris in thesupernatant aspirated. The cell pellet was resuspended in 0.5 ml PBScontaining 0.5% bovine serum albumin (BSA).

The sample was analyzed using a FACS™ brand flow cytometer. Calibrationwas performed using QuantiBRITE™ quantitation kit (BDIS cat. no.340495). A minimum of 2000 monocyte events (CD14⁺ and further delimitedbased on side scatter) were acquired, and PE fluorescence of these cellscharted. FIG. 5 shows the data collected on one such patient sample.

A second aliquot of each sample was fractionated over Ficoll-Hypaque andthe PBMC assayed according to Kox et al., Arch. Intern. Med. 157:389-393(1997); Döcke et al., Nature Med. 3:678-681 (1997); and Docke et al., inSchmitz et al. (eds.), Durchflusszytometrie in der KlinischenZelldiagnostic, Schattauer: Stuttgart and New York (1994), pp. 163-177.

Results for each sample by both methods are plotted in FIG. 6 as filledtriangles. The best-fit linear regression line through the points isshown.

Example 3 Stabilization of CD11b-Specific Fluorescent Signal byChloroquine

Blood from three normal adult donors was collected in standard EDTAVacutainer® tubes and placed directly on ice. Fifty (50) μl of wholeblood was transferred to a tube containing 20 μl of a cocktail of CD11bPE and CD45 PerCP antibodies with or without chloroquine, with thechloroquine-containing tubes having a final chloroquine concentration of3 mM. Blood was stained for the indicated time at room temperaturebefore addition of 1.0 ml FACS lysing reagent (1×). Flow cytometricanalysis was performed essentially as described in Examples 1 and 2.Results are shown in FIG. 7.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in their entiretyas if each had been individually and specifically incorporated byreference herein. While preferred illustrative embodiments of thepresent invention are described, it will be apparent to one skilled inthe art that various changes and modifications may be made thereinwithout departing from the invention, and it is intended in the appendedclaims to cover all such changes and modifications that fall within thetrue spirit and scope of the invention.

1. A composition for flow cytometric measurement of a cell-surfacemolecule on human peripheral blood cells, wherein said cell-surfacemolecule is characterized in that a fluorescent signal measuredfollowing staining with a fluorescently labeled antibody specific forsaid cell-surface molecule in the absence of a lysosomotropic aminedepends on the time of incubation with said antibody, said compositioncomprising: a fluorophore-conjugated antibody specific for saidcell-surface molecule, and a lysosomotropic amine.
 2. The composition ofclaim 1, wherein said lysosomotropic amine is selected from the groupconsisting of chloroquine, hydroxychloroquine, primaquine, andmethylamine.
 3. The composition of claim 2, wherein said lysosomotropicamine is chloroquine.
 4. The composition of claim 2, wherein saidlysosomotropic amine is hydroxychloroquine.
 5. The composition of claim1, wherein said fluorophore is PE.
 6. The composition of claim 5,wherein said PE fluorophore and said antibody are conjugated at adefined molar ratio.
 7. The composition of claim 6, wherein said ratiois 1:1.
 8. A kit for flow cytometric measurement of a cell-surfacemolecule on the surface of peripheral blood cells, comprising: acomposition according to claim 1, and an erythrocyte lysing composition.9. The kit according to claim 8, further comprising: pelletized beadsconjugated with defined levels of PE.